Publications by authors named "Wan-Ting He"

23 Publications

  • Page 1 of 1

Emergence and adaptive evolution of influenza D virus.

Microb Pathog 2021 Sep 15:105193. Epub 2021 Sep 15.

Agricultural College, Jinhua Poletecnic, Jinhua, 321007, China. Electronic address:

As a novel member of the Orthomyxoviridae, influenza D virus (IDV) was firstly isolated from swine. However, cattle were found to serve as its primary reservoir. The study of IDV emergence can shed light into the dynamics of zoonotic infections and interspecies transmission. Although there is an increasing number of strains and sequenced IDV strains, their origin, epidemiology and evolutionary dynamics remain unclear. In this study, we reconstruct the diversity and evolutionary dynamics of IDVs. Molecular detection of swine tissue samples shows that six IDV positive samples were identified in the Eastern China. Phylogenetic analyses suggest three major IDV lineages designated as D/Japan, D/OK and D/660 as well as intermediate lineages. IDVs show strong association with geographical location indicating a high level of local transmission, which suggests IDVs tend to establish a local lineage of in situ evolution. In addition, the D/OK lineage widely circulates in swine in Eastern China, and all of the Chinese virus isolates form a distinct sub-clade (D/China sub-lineage). Furthermore, we identified important amino acids in the HEF gene under positive selection that might affect its receptor binding cavity relevant for its broader cell tropism. The combined results highlight that more attention should be paid to the potential threat of IDV to livestock and farming in China.
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http://dx.doi.org/10.1016/j.micpath.2021.105193DOI Listing
September 2021

Site-Specific Steric Control of SARS-CoV-2 Spike Glycosylation.

Biochemistry 2021 07 2;60(27):2153-2169. Epub 2021 Jul 2.

School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, U.K.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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http://dx.doi.org/10.1021/acs.biochem.1c00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262170PMC
July 2021

A Rapid Assay for SARS-CoV-2 Neutralizing Antibodies That Is Insensitive to Antiretroviral Drugs.

J Immunol 2021 Jun 28. Epub 2021 Jun 28.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA;

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudotyped virus (PSV) assays are widely used to measure neutralization titers of sera and of isolated neutralizing Abs (nAbs). PSV neutralization assays are safer than live virus neutralization assays and do not require access to biosafety level 3 laboratories. However, many PSV assays are nevertheless somewhat challenging and require at least 2 d to carry out. In this study, we report a rapid (<30 min), sensitive, cell-free, off-the-shelf, and accurate assay for receptor binding domain nAb detection. Our proximity-based luciferase assay takes advantage of the fact that the most potent SARS-CoV-2 nAbs function by blocking the binding between SARS-CoV-2 and angiotensin-converting enzyme 2. The method was validated using isolated nAbs and sera from spike-immunized animals and patients with coronavirus disease 2019. The method was particularly useful in patients with HIV taking antiretroviral therapies that interfere with the conventional PSV assay. The method provides a cost-effective and point-of-care alternative to evaluate the potency and breadth of the predominant SARS-CoV-2 nAbs elicited by infection or vaccines.
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http://dx.doi.org/10.4049/jimmunol.2100155DOI Listing
June 2021

Cross-reactive serum and memory B-cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection.

Nat Commun 2021 05 19;12(1):2938. Epub 2021 May 19.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.

Pre-existing immunity to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, induced from natural infection or vaccination. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. Here we compare serum antibody and memory B cell responses to coronavirus spike proteins from pre-pandemic and SARS-CoV-2 convalescent donors using binding and functional assays. We show weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we find evidence of pre-existing cross-reactive memory B cells that are activated during SARS-CoV-2 infection. Monoclonal antibodies show varying degrees of cross-reactivity with betacoronaviruses, including SARS-CoV-1 and endemic coronaviruses. We identify one cross-reactive neutralizing antibody specific to the S2 subunit of the S protein. Our results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.
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http://dx.doi.org/10.1038/s41467-021-23074-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8134462PMC
May 2021

A protective broadly cross-reactive human antibody defines a conserved site of vulnerability on beta-coronavirus spikes.

bioRxiv 2021 Mar 31. Epub 2021 Mar 31.

We recently described CC40.8 bnAb from a COVID-19 donor that exhibits broad reactivity with human β-CoVs. Here, we show that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 Å resolution and found that the peptide adopts a mainly helical structure. Conserved residues in β-CoVs interact with the antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibits protective efficacy against SARS-CoV-2 challenge in a hamster model with reduction in weight loss and lung viral titers. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational vaccine strategies. Overall, our study describes a new target on CoV spikes for protective antibodies that may facilitate the development of pan-β-CoV vaccines.

Summary: A human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope promising for pan-β-CoV vaccine strategies.
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http://dx.doi.org/10.1101/2021.03.30.437769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020973PMC
March 2021

Site-specific steric control of SARS-CoV-2 spike glycosylation.

bioRxiv 2021 Mar 9. Epub 2021 Mar 9.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.
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http://dx.doi.org/10.1101/2021.03.08.433764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986994PMC
March 2021

Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection.

bioRxiv 2020 Sep 23. Epub 2020 Sep 23.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.
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http://dx.doi.org/10.1101/2020.09.22.308965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523096PMC
September 2020

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model.

Science 2020 08 15;369(6506):956-963. Epub 2020 Jun 15.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained weight and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.
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http://dx.doi.org/10.1126/science.abc7520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299280PMC
August 2020

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model.

bioRxiv 2020 May 15. Epub 2020 May 15.

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.
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http://dx.doi.org/10.1101/2020.05.11.088674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263516PMC
May 2020

Development of a Man-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses.

Virulence 2020 01;11(1):707-718

MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University , Nanjing, China.

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order . Here, we developed a multiplex Man-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and Man fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 10 copies/μL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.
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http://dx.doi.org/10.1080/21505594.2020.1771980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549975PMC
January 2020

Genomic Epidemiology, Evolution, and Transmission Dynamics of Porcine Deltacoronavirus.

Mol Biol Evol 2020 09;37(9):2641-2654

MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, Jiangsu Engineering Laboratory of Animal Immunology, Institute of Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown once again that coronavirus (CoV) in animals are potential sources for epidemics in humans. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen of swine with a worldwide distribution. Here, we implemented and described an approach to analyze the epidemiology of PDCoV following its emergence in the pig population. We performed an integrated analysis of full genome sequence data from 21 newly sequenced viruses, along with comprehensive epidemiological surveillance data collected globally over the last 15 years. We found four distinct phylogenetic lineages of PDCoV, which differ in their geographic circulation patterns. Interestingly, we identified more frequent intra- and interlineage recombination and higher virus genetic diversity in the Chinese lineages compared with the USA lineage where pigs are raised in different farming systems and ecological environments. Most recombination breakpoints are located in the ORF1ab gene rather than in genes encoding structural proteins. We also identified five amino acids under positive selection in the spike protein suggesting a role for adaptive evolution. According to structural mapping, three positively selected sites are located in the N-terminal domain of the S1 subunit, which is the most likely involved in binding to a carbohydrate receptor, whereas the other two are located in or near the fusion peptide of the S2 subunit and thus might affect membrane fusion. Finally, our phylogeographic investigations highlighted notable South-North transmission as well as frequent long-distance dispersal events in China that could implicate human-mediated transmission. Our findings provide new insights into the evolution and dispersal of PDCoV that contribute to our understanding of the critical factors involved in CoVs emergence.
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http://dx.doi.org/10.1093/molbev/msaa117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454817PMC
September 2020

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Binding to ACE2 Receptors from Human, Pets, Farm Animals, and Putative Intermediate Hosts.

J Virol 2020 07 16;94(15). Epub 2020 Jul 16.

Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

The emergence of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in a pandemic. Here, we used X-ray structures of human ACE2 bound to the receptor-binding domain (RBD) of the spike protein (S) from SARS-CoV-2 to predict its binding to ACE2 proteins from different animals, including pets, farm animals, and putative intermediate hosts of SARS-CoV-2. Comparing the interaction sites of ACE2 proteins known to serve or not serve as receptors allows the definition of residues important for binding. From the 20 amino acids in ACE2 that contact S, up to 7 can be replaced and ACE2 can still function as the SARS-CoV-2 receptor. These variable amino acids are clustered at certain positions, mostly at the periphery of the binding site, while changes of the invariable residues prevent S binding or infection of the respective animal. Some ACE2 proteins even tolerate the loss or acquisition of N-glycosylation sites located near the S interface. Of note, pigs and dogs, which are not infected or are not effectively infected and have only a few changes in the binding site, exhibit relatively low levels of ACE2 in the respiratory tract. Comparison of the RBD of S of SARS-CoV-2 with that from bat coronavirus strain RaTG13 (Bat-CoV-RaTG13) and pangolin coronavirus (Pangolin-CoV) strain hCoV-19/pangolin/Guangdong/1/2019 revealed that the latter contains only one substitution, whereas Bat-CoV-RaTG13 exhibits five. However, ACE2 of pangolin exhibits seven changes relative to human ACE2, and a similar number of substitutions is present in ACE2 of bats, raccoon dogs, and civets, suggesting that SARS-CoV-2 may not be especially adapted to ACE2 of any of its putative intermediate hosts. These analyses provide new insight into the receptor usage and animal source/origin of SARS-CoV-2. SARS-CoV-2 is threatening people worldwide, and there are no drugs or vaccines available to mitigate its spread. The origin of the virus is still unclear, and whether pets and livestock can be infected and transmit SARS-CoV-2 are important and unknown scientific questions. Effective binding to the host receptor ACE2 is the first prerequisite for infection of cells and determines the host range. Our analysis provides a framework for the prediction of potential hosts of SARS-CoV-2. We found that ACE2 from species known to support SARS-CoV-2 infection tolerate many amino acid changes, indicating that the species barrier might be low. Exceptions are dogs and especially pigs, which revealed relatively low ACE2 expression levels in the respiratory tract. Monitoring of animals is necessary to prevent the generation of a new coronavirus reservoir. Finally, our analysis also showed that SARS-CoV-2 may not be specifically adapted to any of its putative intermediate hosts.
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http://dx.doi.org/10.1128/JVI.00831-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375388PMC
July 2020

COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives.

Trends Mol Med 2020 05 21;26(5):483-495. Epub 2020 Mar 21.

MOE Joint International Research Laboratory of Animal Health and Food Safety, Jiangsu Engineering Laboratory of Animal Immunology, Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. Electronic address:

The recent outbreak of COVID-19 in Wuhan turned into a public health emergency of international concern. With no antiviral drugs nor vaccines, and the presence of carriers without obvious symptoms, traditional public health intervention measures are significantly less effective. Here, we report the epidemiological and virological characteristics of the COVID-19 outbreak. Originated in bats, 2019-nCoV/ severe acute respiratory syndrome coronavirus (SARS-CoV)-2 likely experienced adaptive evolution in intermediate hosts before transfer to humans at a concentrated source of transmission. Similarities of receptor sequence binding to 2019-nCoV between humans and animals suggest a low species barrier for transmission of the virus to farm animals. We propose, based on the One Health model, that veterinarians and animal specialists should be involved in a cross-disciplinary collaboration in the fight against this epidemic.
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http://dx.doi.org/10.1016/j.molmed.2020.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118693PMC
May 2020

Efficient quantum key distribution against collective noise using polarization and transverse spatial mode of photons.

Opt Express 2020 Feb;28(4):4611-4624

Channel noise is the main issue which reduces the efficiency of quantum communication. Here we present an efficient scheme for quantum key distribution against collective-rotation channel noise using polarization and transverse spatial mode of photons. Exploiting the two single-photon Bell states and two-photon hyperentangled Bell states in the polarization and the transverse spatial mode degrees of freedom (DOFs), the mutually unbiased bases can be encoded for logical qubits against the collective-rotation noise. Our scheme shows noiseless subspaces can be made up of two DOFs of two photons instead of multiple photons, which will reduce the resources required for noiseless subspaces and depress the photonic loss sensitivity. Moreover, the two single-photon Bell states and two-photon hyperentangled Bell states are symmetrical to the two photons, which means the relative order of the two photons is not required in our scheme, so the receiver only needs to measure the state of each photon, which makes our protocol easy to execute in experiment than the previous works.
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http://dx.doi.org/10.1364/OE.374292DOI Listing
February 2020

Adaption and parallel evolution of human-isolated H5 avian influenza viruses.

J Infect 2020 06 31;80(6):630-638. Epub 2020 Jan 31.

MOE International Joint Collaborative Research Laboratory for Animal Health & Food Safety, Jiangsu Engineering Laboratory of Animal Immunology, Institute of Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. Electronic address:

Avian-to-human transmission of highly pathogenic avian influenza viruses (HPAIV) and their subsequent adaptation to humans are of great concern to public health. Surveillance and early warning of AIVs with the potential to infect humans and pandemic potential is crucial. In this study, we determined whether adaptive evolution occurred in human-isolated H5 viruses. We evaluated all available genomes of H5N1 and H5N6 avian influenza A virus. Firstly, we systematically identified several new mutations in H5 AIV that might be associated with human adaptation using a combination of novel comparative phylogenetic methods and structural analysis. Some changes are the result of parallel evolution, further demonstrating their importance. In total, we identified 102 adaptive evolution sites in eight genes. Some residues had been previously identified, such as 227 in HA and 627 in PB2, while others have not been reported so far. Ten sites from four genes evolved in parallel but no obvious positive selection was detected. Our study suggests that during infection of humans, H5 viruses evolved to adapt to their new host environment and that the sites of adaptive/parallel evolution might play a role in crossing the species barrier and are the response to new selection pressure. The results provide insight to implement early detection systems for transitional stages in H5 AIV evolution before its potential adaptation for humans. Author summary line The prerequisite of surveillance and early warning of avian influenza viruses with the potential to infect humans depends on the identification of human-adaptation related mutations. In this study, we used a novel approach combining both phylogenetic and structural analysis to identify possible human-adaptation related mutations in H5 AIVs. Previous studies reported human-adaptation related mutations and some novel mutations exhibiting parallel evolution. Our result provides new insights into how AIVs adapt to humans by point mutations.
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http://dx.doi.org/10.1016/j.jinf.2020.01.012DOI Listing
June 2020

[Clinical observation on the therapeutic effect of warm acupuncture on endometrial cavity fluid from in vitro fertilization-embryo transfer].

Zhongguo Zhen Jiu 2019 Sep;39(9):923-6

Department of TCM, Foshan Woman and Children Hospital, Foshan 52800, Guangdong Province, China.

Objective: To observe the clinical effect of warm acupuncture on endometrial cavity fluid (ECF) from in vitro fertilization-embryo transfer (IVF-ET), and to explore the mechanism of warm acupuncture on ECF.

Methods: Twenty-nine patients who were prepared for IVF-ET from 2016 to 2019 and whose transplantation was cancelled due to ECF found by vaginal B-ultrasound examination were divided into an observation group (14 cases) and a control group (15 cases) according to random number table method. The warm acupuncture was applied at Zhongwan (CV 12), Qihai (CV 6), Guanyuan (CV 4), Zhongji (CV 3), Guilai (ST 29), Zigong (EX-CA 1), Zusanli (ST 36), Sanyinjiao (SP 6) after the end of menstruation in the observation group, the treatment lasted for 60 min, once a day, 5 times as a course, with 2 days interval between the courses and 3 consecutive courses of treatment were given, until the embryo transfer was performed in the IVF assisted pregnancy cycle. After the end of menstruation, fresh leonurus japonicus capsule was given in the control group, 3 times a day, 0.8 g each time, 7 days as a course, and 3 courses of continuous treatment were received, until the embryo transfer was performed in the IVF assisted pregnancy cycle. The changes of ECF before and after treatment, the time required to prepare for embryo transfer during IVF assisted pregnancy cycle, and the clinical outcome of embryo transfer were observed in the two groups.

Results: The decrease of ECF in the observation group was more significant than that in the control group (<0.05). The time required for the embryo transfer in the IVF assisted pregnancy cycle in the observation group was shorter than that in the control group (<0.05). The clinical pregnancy rate in the observation group was 42.9% (6/14), which was significantly higher than 26.7% (4/15) in the control group (<0.05).

Conclusion: Warm acupuncture may improve the clinical pregnancy rate by raising the local temperature of the lower abdomen, accelerating the blood circulation around the uterus and appendages, promoting the absorption of ECF, improving the uterine environment and endometrial receptivity.
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http://dx.doi.org/10.13703/j.0255-2930.2019.09.003DOI Listing
September 2019

Protein kinase D at the Golgi controls NLRP3 inflammasome activation.

J Exp Med 2017 Sep 17;214(9):2671-2693. Epub 2017 Jul 17.

Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France

The inflammasomes are multiprotein complexes sensing tissue damage and infectious agents to initiate innate immune responses. Different inflammasomes containing distinct sensor molecules exist. The NLRP3 inflammasome is unique as it detects a variety of danger signals. It has been reported that NLRP3 is recruited to mitochondria-associated endoplasmic reticulum membranes (MAMs) and is activated by MAM-derived effectors. Here, we show that in response to inflammasome activators, MAMs localize adjacent to Golgi membranes. Diacylglycerol (DAG) at the Golgi rapidly increases, recruiting protein kinase D (PKD), a key effector of DAG. Upon PKD inactivation, self-oligomerized NLRP3 is retained at MAMs adjacent to Golgi, blocking assembly of the active inflammasome. Importantly, phosphorylation of NLRP3 by PKD at the Golgi is sufficient to release NLRP3 from MAMs, resulting in assembly of the active inflammasome. Moreover, PKD inhibition prevents inflammasome autoactivation in peripheral blood mononuclear cells from patients carrying NLRP3 mutations. Hence, Golgi-mediated PKD signaling is required and sufficient for NLRP3 inflammasome activation.
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http://dx.doi.org/10.1084/jem.20162040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584123PMC
September 2017

The MLKL Channel in Necroptosis Is an Octamer Formed by Tetramers in a Dyadic Process.

Mol Cell Biol 2017 03 15;37(5). Epub 2017 Feb 15.

State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, China

Oligomerization of the mixed-lineage kinase domain-like protein (MLKL) is essential for its cation channel function in necroptosis. Here we show that the MLKL channel is an octamer comprising two previously identified tetramers most likely in their side-by-side position. Intermolecule disulfide bonds are present in the tetramer but are not required for octamer assembly and necroptosis. MLKL forms oligomers in the necrosome and is then released from the necrosome before or during its membrane translocation. We identified two MLKL mutants that could not oligomerize into octamers, although they formed a tetramer, and also, one MLKL mutant could spontaneously form a disulfide bond-linked octamer. Subsequent analysis revealed that the tetramers fail to translocate to the plasma membrane and that the MLKL octamer formation depends on α-helices 4 and 5. While MLKL could be detected from outside the cells, its N- and C-terminal ends could not be detected, indicating that the MLKL octamer spans across the plasma membrane, leaving its N and C termini inside the cell. These data allowed us to propose a 180° symmetry model of the MLKL octamer and conclude that the fully assembled MLKL octamers, but not the previously described tetramers, act as effectors of necroptosis.
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http://dx.doi.org/10.1128/MCB.00497-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311246PMC
March 2017

Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis.

Cell Res 2016 09 30;26(9):1007-20. Epub 2016 Aug 30.

State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

Necroptosis and pyroptosis are two forms of programmed cell death with a common feature of plasma membrane rupture. Here we studied the morphology and mechanism of pyroptosis in comparison with necroptosis. Different from necroptosis, pyroptosis undergoes membrane blebbing and produces apoptotic body-like cell protrusions (termed pyroptotic bodies) prior to plasma membrane rupture. The rupture in necroptosis is explosion-like, whereas in pyroptosis it leads to flattening of cells. It is known that the execution of necroptosis is mediated by mixed lineage kinase domain-like (MLKL) oligomers in the plasma membrane, whereas gasdermin-D (GSDMD) mediates pyroptosis after its cleavage by caspase-1 or caspase-11. We show that N-terminal fragment of GSDMD (GSDMD-N) generated by caspase cleavage also forms oligomer and migrates to the plasma membrane to kill cells. Both MLKL and GSDMD-N are lipophilic and the N-terminal sequences of both proteins are important for their oligomerization and plasma membrane translocation. Unlike MLKL which forms channels on the plasma membrane that induces influx of selected ions which osmotically swell the cells to burst, GSDMD-N forms non-selective pores and does not rely on increased osmolarity to disrupt cells. Our study reveals the pore-forming activity of GSDMD and channel-forming activity of MLKL determine different ways of plasma membrane rupture in pyroptosis and necroptosis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034106PMC
http://dx.doi.org/10.1038/cr.2016.100DOI Listing
September 2016

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.

Cell Res 2015 Dec 27;25(12):1285-98. Epub 2015 Nov 27.

State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1β (IL-1β) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1β in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1β secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd(-/-) cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd(-/-) cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.
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http://dx.doi.org/10.1038/cr.2015.139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670995PMC
December 2015

[Relation of intermediate-conductance Ca(2+)-activated K(+) channels with ability of proliferation, migration, invasion and IgE secretion of multiple myeloma cells].

Zhongguo Shi Yan Xue Ye Xue Za Zhi 2014 Jun;22(3):742-6

Department of Haematology, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, Heilongjian Province, China.

This study was aimed to investigate the effects of the intermediate-conductance Ca(2+)-activated K(+) (IKCa1) channels on the proliferation, migration, invasion ability and monoclonal immunoglobulin (IgE) secretion of multiple myeloma (MM) cells. Trypan blue exclusion was used to evaluate the impact of clotrimazole (CLO, an inhibitor of the KCa1) on the survival ability of MM cell line U266; transwell chamber and matrigel experiments were used to evaluate the impact of CLO on the ability of the migration and invasion of U266 cells; the influence of CLO on IgE secretion in U266 cells was detected by ELISA. The results showed that small dose of CLO ( ≤ 1.0 µmol/L) could not inhibit the viability of U266 cells. The Transwell and Matrigel invading tests displayed that the cell number moving into lower chamber of transwell decreased after U266 cells treated with small dose of CLO ( ≤ 1.0 µmol/L). After treating the cells with 1.00 µmol/L CLO for 24 h and 48 h, the concentration of IgE in cell supernatant was (4.98 ± 0.39) and (4.38 ± 0.32) ng/ml, while those in control group were (15.41 ± 1.88) and (19.73 ± 2.01) ng/ml, respectively, suggesting significant difference between them(P < 0.05). It is concluded that CLO can decrease the ability of migration and monoclonal immunoglobulin secretion of multiple myeloma cells by blocking the IKCa1, thus this study provides a new think for the targeted therapy of MM.
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http://dx.doi.org/10.7534/j.issn.1009-2137.2014.03.031DOI Listing
June 2014

pelo is required for high efficiency viral replication.

PLoS Pathog 2014 Apr 10;10(4):e1004034. Epub 2014 Apr 10.

State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian, China.

Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection.
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http://dx.doi.org/10.1371/journal.ppat.1004034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983054PMC
April 2014

Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death.

Cell Res 2014 Jan 24;24(1):105-21. Epub 2013 Dec 24.

State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.

Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture.
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http://dx.doi.org/10.1038/cr.2013.171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879712PMC
January 2014
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